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Blois S, Goetz BM, Mojumder A, Sullivan CS. Shedding dynamics of a DNA virus population during acute and long-term persistent infection. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.31.646279. [PMID: 40236044 PMCID: PMC11996411 DOI: 10.1101/2025.03.31.646279] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/17/2025]
Abstract
Although much is known of the molecular mechanisms of virus infection within cells, substantially less is understood about within-host infection. Such knowledge is key to understanding how viruses take up residence and transmit infectious virus, in some cases throughout the life of the host. Here, using murine polyomavirus (muPyV) as a tractable model, we monitor parallel infections of thousands of differentially barcoded viruses within a single host. In individual mice, we show that numerous viruses (>2600) establish infection and are maintained for long periods post-infection. Strikingly, a low level of many different barcodes is shed in urine at all times post-infection, with a minimum of at least 80 different barcodes present in every sample throughout months of infection. During the early acute phase, bulk shed virus genomes derive from numerous different barcodes. This is followed by long term persistent infection detectable in diverse organs. Consistent with limited productive exchange of virus genomes between organs, each displays a unique pattern of relative barcode abundance. During the persistent phase, constant low-level shedding of typically hundreds of barcodes is maintained but is overlapped with rare, punctuated shedding of high amounts of one or a few individual barcodes. In contrast to the early acute phase, these few infrequent highly shed barcodes comprise the majority of bulk shed genomes observed during late times of persistent infection, contributing to a stark decrease in bulk barcode diversity that is shed over time. These temporally shifting patterns, which are conserved across hosts, suggest that polyomaviruses balance continuous transmission potential with reservoir-driven high-level reactivation. This offers a mechanistic basis for polyomavirus ubiquity and long-term persistence, which are typical of many DNA viruses. Author Summary / Importance Polyomavirus infections, mostly benign but potentially fatal for immunocompromised individuals, undergo acute and long-term persistent infections. Typically, polyomavirus-associated diseases arise due to virus infection occurring in the context of a persistently infected individual. However, little is understood regarding the mechanisms of how polyomaviruses establish, maintain, and reactivate from persistent infection. We developed a non-invasive virus shedding assay combining barcoded murine polyomavirus, massively parallel sequencing technology, and novel computational approaches to track long-term infections in mice. We expect these methods to be of use not only to the study of DNA viruses but also for understanding persitent infection of diverse microbes. The study revealed organ-specific virus reservoirs and two distinct shedding patterns: constant low-level shedding of numerous barcodes and episodic high-level shedding of few barcodes. Over time, the diversity of shed barcodes decreased substantially. These findings suggest a persistent low-level infection in multiple reservoirs, with occasional bursts of replication in a small subset of infected cells. This combination of broad reservoirs and varied shedding mechanisms may contribute to polyomavirus success in transmission and maintaining long-term infections.
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Saribas AS, Jensen LE, Safak M. Recent advances in discovery and functional analysis of the small proteins and microRNA expressed by polyomaviruses. Virology 2025; 602:110310. [PMID: 39612622 DOI: 10.1016/j.virol.2024.110310] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2024] [Revised: 11/13/2024] [Accepted: 11/18/2024] [Indexed: 12/01/2024]
Abstract
The polyomavirus family consists of a highly diverse group of small DNA viruses isolated from various species, including humans. Some family members have been used as model systems to understand the fundamentals of modern biology. After the discovery of the first two human polyomaviruses (JC virus and BK virus) during the early 1970s, their current number reached 14 today. Some family members cause considerably severe human diseases, including polyomavirus-associated nephropathy (PVAN), progressive multifocal leukoencephalopathy (PML), trichodysplasia spinulosa (TS) and Merkel cell carcinoma (MCC). Polyomaviruses encode universal regulatory and structural proteins, but some members express additional virus-specific proteins and microRNA, which significantly contribute to the viral biology, cell transformation, and perhaps progression of the disease that they are associated with. In the current review, we summarized the recent advances in discovery, and functional and structural analysis of those viral proteins and microRNA.
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Affiliation(s)
- A Sami Saribas
- Lewis Katz School of Medicine at Temple University, Department of Microbiology, Immunology and Inflammation Center for Neurovirology and Gene Editing, 3500 N. Broad Street, Philadelphia, PA, 19140, USA.
| | - Liselotte E Jensen
- Lewis Katz School of Medicine at Temple University, Department of Microbiology, Immunology and Inflammation, Center for Inflammation and Lung Research, 3500 N. Broad Street, Philadelphia, PA, 19140, USA
| | - Mahmut Safak
- Lewis Katz School of Medicine at Temple University, Department of Microbiology, Immunology and Inflammation Center for Neurovirology and Gene Editing, 3500 N. Broad Street, Philadelphia, PA, 19140, USA.
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Vasiliūnaitė E, Repšytė M, Kramer EM, Lang J, Jelinek C, Ulrich RG, Buck CB, Gedvilaitė A. Novel polyomavirus in the endangered garden dormouse Eliomys quercinus. Virol J 2024; 21:309. [PMID: 39605065 PMCID: PMC11603729 DOI: 10.1186/s12985-024-02581-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2024] [Accepted: 11/14/2024] [Indexed: 11/29/2024] Open
Abstract
BACKGROUND The garden dormouse (Eliomys quercinus) has experienced a significant population decline across Europe in recent decades. While habitat loss and climate change are often cited as primary factors, pathogen exposure, either to novel or to previously known, may play a role in such a decline. This study aimed to investigate the presence of polyomaviruses in garden dormice, given that these viruses are highly prevalent and can cause disease, particularly in immunocompromised individuals. METHODS The carcasses of garden dormice (n = 89) were collected throughout Germany. Kidney samples were tested for the presence of polyomavirus DNA using nested degenerate and specific diagnostic PCRs. Seroprevalence was assessed from chest cavity fluid samples through an enzyme-linked immunosorbent assay using polyomavirus VP1 virus-like particles produced in yeast. RESULTS A new polyomavirus, related to chimpanzee (Pan troglodytes) polyomaviruses 4 and 5 and human Merkel cell polyomavirus, was identified in the garden dormouse. Two 5,380 bp-length complete viral genomes were sequenced from dormice kidney samples (sequences PQ246041 and PQ246042). Genes encoding the putative structural proteins VP1, VP2, and VP3, as well as the Large, Middle, and small T antigens, containing conserved functional domains were identified. Polyomavirus DNA was detected in 2 of 74 dormice (2.7%, 95% confidence interval: 0-6.4%) through PCR, while 12 of 69 animals (17.4%, 95% confidence interval: 8.4-26.3%) tested positive for polyomavirus-specific antibodies. CONCLUSIONS In conclusion, here we describe a novel polyomavirus in the garden dormouse with molecular and serological detection. Pairwise sequence comparison and phylogenetic analysis suggest that this novel virus may represent a novel species within the genus Alphapolyomavirus. Future work should examine if this virus is garden dormouse-specific and whether it is associated with disease in dormice.
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Affiliation(s)
- Emilija Vasiliūnaitė
- Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio al. 7, Vilnius, LT-10257, Lithuania.
| | - Monika Repšytė
- Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio al. 7, Vilnius, LT-10257, Lithuania
| | - Eva Marie Kramer
- Clinic for Birds, Reptiles, Amphibians and Fish, Working Group for Wildlife Research, Justus-Liebig-University Gießen, Frankfurter Strasse 104, D-35392, Gießen, Germany
| | - Johannes Lang
- Clinic for Birds, Reptiles, Amphibians and Fish, Working Group for Wildlife Research, Justus-Liebig-University Gießen, Frankfurter Strasse 104, D-35392, Gießen, Germany
| | - Christine Jelinek
- Clinic for Birds, Reptiles, Amphibians and Fish, Working Group for Wildlife Research, Justus-Liebig-University Gießen, Frankfurter Strasse 104, D-35392, Gießen, Germany
| | - Rainer G Ulrich
- Friedrich-Loeffler-Institut (FLI) Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Gießen, Germany
| | - Christopher B Buck
- Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, 20892-4263, USA
| | - Alma Gedvilaitė
- Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio al. 7, Vilnius, LT-10257, Lithuania
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Peng WY, Abere B, Shi H, Toland S, Smithgall TE, Moore PS, Chang Y. Membrane-bound Merkel cell polyomavirus middle T protein constitutively activates PLCγ1 signaling through Src-family kinases. Proc Natl Acad Sci U S A 2023; 120:e2316467120. [PMID: 38079542 PMCID: PMC10740393 DOI: 10.1073/pnas.2316467120] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2023] [Accepted: 11/11/2023] [Indexed: 12/18/2023] Open
Abstract
Merkel cell polyomavirus (MCV or MCPyV) is an alphapolyomavirus causing human Merkel cell carcinoma and encodes four tumor (T) antigen proteins: large T (LT), small tumor (sT), 57 kT, and middle T (MT)/alternate LT open reading frame proteins. We show that MCV MT is generated as multiple isoforms through internal methionine translational initiation that insert into membrane lipid rafts. The membrane-localized MCV MT oligomerizes and promiscuously binds to lipid raft-associated Src family kinases (SFKs). MCV MT-SFK interaction is mediated by a Src homology (SH) 3 recognition motif as determined by surface plasmon resonance, coimmunoprecipitation, and bimolecular fluorescence complementation assays. SFK recruitment by MT leads to tyrosine phosphorylation at a SH2 recognition motif (pMTY114), allowing interaction with phospholipase C gamma 1 (PLCγ1). The secondary recruitment of PLCγ1 to the SFK-MT membrane complex promotes PLCγ1 tyrosine phosphorylation on Y783 and activates the NF-κB inflammatory signaling pathway. Mutations at either the MCV MT SH2 or SH3 recognition sites abrogate PLCγ1-dependent activation of NF-κB signaling and increase viral replication after MCV genome transfection into 293 cells. These findings reveal a conserved viral targeting of the SFK-PLCγ1 pathway by both MCV and murine polyomavirus (MuPyV) MT proteins. The molecular steps in how SFK-PLCγ1 activation is achieved, however, differ between these two viruses.
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Affiliation(s)
- Wen-Yu Peng
- School of Medicine, Tsinghua University, Beijing100084, China
- Cancer Virology Program, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA15213
| | - Bizunesh Abere
- Cancer Virology Program, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA15213
- Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA15219
| | - Haibin Shi
- Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA15219
| | - Sabrina Toland
- Cancer Virology Program, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA15213
| | - Thomas E. Smithgall
- Cancer Virology Program, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA15213
- Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA15219
| | - Patrick S. Moore
- Cancer Virology Program, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA15213
- Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA15219
| | - Yuan Chang
- Cancer Virology Program, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA15213
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA15213
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Jayatilleke KM, Duivenvoorden HM, Ryan GF, Parker BS, Hulett MD. Investigating the Role of Heparanase in Breast Cancer Development Utilising the MMTV-PyMT Murine Model of Mammary Carcinoma. Cancers (Basel) 2023; 15:cancers15113062. [PMID: 37297024 DOI: 10.3390/cancers15113062] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Revised: 05/30/2023] [Accepted: 05/31/2023] [Indexed: 06/12/2023] Open
Abstract
Breast cancer is the second most common human malignancy and is a major global health burden. Heparanase (HPSE) has been widely implicated in enhancing the development and progression of solid tumours, including breast cancer. In this study, the well-established spontaneous mammary tumour-developing MMTV-PyMT murine model was utilised to examine the role of HPSE in breast cancer establishment, progression, and metastasis. The use of HPSE-deficient MMTV-PyMT (MMTV-PyMTxHPSE-/-) mice addressed the lack of genetic ablation models to investigate the role of HPSE in mammary tumours. It was demonstrated that even though HPSE regulated mammary tumour angiogenesis, mammary tumour progression and metastasis were HPSE-independent. Furthermore, there was no evidence of compensatory action by matrix metalloproteinases (MMPs) in response to the lack of HPSE expression in the mammary tumours. These findings suggest that HPSE may not play a significant role in the mammary tumour development of MMTV-PyMT animals. Collectively, these observations may have implications in the clinical setting of breast cancer and therapy using HPSE inhibitors.
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Affiliation(s)
- Krishnath M Jayatilleke
- Department of Biochemistry and Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia
| | - Hendrika M Duivenvoorden
- Department of Biochemistry and Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia
- School of Biological Sciences, Monash University, Clayton, VIC 3168, Australia
| | - Gemma F Ryan
- Department of Biochemistry and Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia
| | - Belinda S Parker
- Department of Biochemistry and Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia
- Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3052, Australia
| | - Mark D Hulett
- Department of Biochemistry and Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia
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Janovec V, Ryabchenko B, Škarková A, Pokorná K, Rösel D, Brábek J, Weber J, Forstová J, Hirsch I, Huérfano S. TLR4-Mediated Recognition of Mouse Polyomavirus Promotes Cancer-Associated Fibroblast-Like Phenotype and Cell Invasiveness. Cancers (Basel) 2021; 13:cancers13092076. [PMID: 33923020 PMCID: PMC8123340 DOI: 10.3390/cancers13092076] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2021] [Revised: 04/15/2021] [Accepted: 04/21/2021] [Indexed: 11/16/2022] Open
Abstract
The tumorigenic potential of mouse polyomavirus (MPyV) has been studied for decades in cell culture models and has been mainly attributed to nonstructural middle T antigen (MT), which acts as a scaffold signal adaptor, activates Src tyrosine kinases, and possesses transforming ability. We hypothesized that MPyV could also transform mouse cells independent of MT via a Toll-like receptor 4 (TLR4)-mediated inflammatory mechanism. To this end, we investigated the interaction of MPyV with TLR4 in mouse embryonic fibroblasts (MEFs) and 3T6 cells, resulting in secretion of interleukin 6 (IL-6), independent of active viral replication. TLR4 colocalized with MPyV capsid protein VP1 in MEFs. Neither TLR4 activation nor recombinant IL-6 inhibited MPyV replication in MEFs and 3T6 cells. MPyV induced STAT3 phosphorylation through both direct and MT-dependent and indirect and TLR4/IL-6-dependent mechanisms. We demonstrate that uninfected mouse fibroblasts exposed to the cytokine environment from MPyV-infected fibroblasts upregulated the expressions of MCP-1, CCL-5, and α-SMA. Moreover, the cytokine microenvironment increased the invasiveness of MEFs and CT26 carcinoma cells. Collectively, TLR4 recognition of MPyV induces a cytokine environment that promotes the cancer-associated fibroblast (CAF)-like phenotype in noninfected fibroblasts and increases cell invasiveness.
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Affiliation(s)
- Vaclav Janovec
- Department of Genetics and Microbiology, Faculty of Science, Charles University, BIOCEV, 25150 Vestec, Czech Republic; (V.J.); (B.R.); (J.F.); (S.H.)
- IOCB Gilead Research Center, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 16000 Prague, Czech Republic; (K.P.); (J.W.)
| | - Boris Ryabchenko
- Department of Genetics and Microbiology, Faculty of Science, Charles University, BIOCEV, 25150 Vestec, Czech Republic; (V.J.); (B.R.); (J.F.); (S.H.)
| | - Aneta Škarková
- Department of Cell Biology, Faculty of Science, Charles University, BIOCEV, 25150 Vestec, Czech Republic; (A.Š.); (D.R.); (J.B.)
| | - Karolína Pokorná
- IOCB Gilead Research Center, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 16000 Prague, Czech Republic; (K.P.); (J.W.)
| | - Daniel Rösel
- Department of Cell Biology, Faculty of Science, Charles University, BIOCEV, 25150 Vestec, Czech Republic; (A.Š.); (D.R.); (J.B.)
| | - Jan Brábek
- Department of Cell Biology, Faculty of Science, Charles University, BIOCEV, 25150 Vestec, Czech Republic; (A.Š.); (D.R.); (J.B.)
| | - Jan Weber
- IOCB Gilead Research Center, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 16000 Prague, Czech Republic; (K.P.); (J.W.)
| | - Jitka Forstová
- Department of Genetics and Microbiology, Faculty of Science, Charles University, BIOCEV, 25150 Vestec, Czech Republic; (V.J.); (B.R.); (J.F.); (S.H.)
| | - Ivan Hirsch
- Department of Genetics and Microbiology, Faculty of Science, Charles University, BIOCEV, 25150 Vestec, Czech Republic; (V.J.); (B.R.); (J.F.); (S.H.)
- IOCB Gilead Research Center, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 16000 Prague, Czech Republic; (K.P.); (J.W.)
- Institute of Molecular Genetics of the Czech Academy of Sciences, 14220 Prague, Czech Republic
- Correspondence: ; Tel.: +420-221-951-723
| | - Sandra Huérfano
- Department of Genetics and Microbiology, Faculty of Science, Charles University, BIOCEV, 25150 Vestec, Czech Republic; (V.J.); (B.R.); (J.F.); (S.H.)
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Peters DK, Erickson KD, Garcea RL. Live Cell Microscopy of Murine Polyomavirus Subnuclear Replication Centers. Viruses 2020; 12:v12101123. [PMID: 33023278 PMCID: PMC7650712 DOI: 10.3390/v12101123] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2020] [Revised: 09/22/2020] [Accepted: 09/30/2020] [Indexed: 01/24/2023] Open
Abstract
During polyomavirus (PyV) infection, host proteins localize to subnuclear domains, termed viral replication centers (VRCs), to mediate viral genome replication. Although the protein composition and spatial organization of VRCs have been described using high-resolution immunofluorescence microscopy, little is known about the temporal dynamics of VRC formation over the course of infection. We used live cell fluorescence microscopy to analyze VRC formation during murine PyV (MuPyV) infection of a mouse fibroblast cell line that constitutively expresses a GFP-tagged replication protein A complex subunit (GFP-RPA32). The RPA complex forms a heterotrimer (RPA70/32/14) that regulates cellular DNA replication and repair and is a known VRC component. We validated previous observations that GFP-RPA32 relocalized to sites of cellular DNA damage in uninfected cells and to VRCs in MuPyV-infected cells. We then used GFP-RPA32 as a marker of VRC formation and expansion during live cell microscopy of infected cells. VRC formation occurred at variable times post-infection, but the rate of VRC expansion was similar between cells. Additionally, we found that the early viral protein, small TAg (ST), was required for VRC expansion but not VRC formation, consistent with the role of ST in promoting efficient vDNA replication. These results demonstrate the dynamic nature of VRCs over the course of infection and establish an approach for analyzing viral replication in live cells.
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Affiliation(s)
- Douglas K. Peters
- BioFrontiers Institute, University of Colorado Boulder, Boulder, CO 80309, USA; (D.K.P.); (K.D.E.)
| | - Kimberly D. Erickson
- BioFrontiers Institute, University of Colorado Boulder, Boulder, CO 80309, USA; (D.K.P.); (K.D.E.)
| | - Robert L. Garcea
- BioFrontiers Institute, University of Colorado Boulder, Boulder, CO 80309, USA; (D.K.P.); (K.D.E.)
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, Boulder, CO 80309, USA
- Correspondence:
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Peters DK, Garcea RL. Murine polyomavirus DNA transitions through spatially distinct nuclear replication subdomains during infection. PLoS Pathog 2020; 16:e1008403. [PMID: 32203554 PMCID: PMC7117779 DOI: 10.1371/journal.ppat.1008403] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2019] [Revised: 04/02/2020] [Accepted: 02/13/2020] [Indexed: 12/16/2022] Open
Abstract
The replication of small DNA viruses requires both host DNA replication and repair factors that are often recruited to subnuclear domains termed viral replication centers (VRCs). Aside from serving as a spatial focus for viral replication, little is known about these dynamic areas in the nucleus. We investigated the organization and function of VRCs during murine polyomavirus (MuPyV) infection using 3D structured illumination microscopy (3D-SIM). We localized MuPyV replication center components, such as the viral large T-antigen (LT) and the cellular replication protein A (RPA), to spatially distinct subdomains within VRCs. We found that viral DNA (vDNA) trafficked sequentially through these subdomains post-synthesis, suggesting their distinct functional roles in vDNA processing. Additionally, we observed disruption of VRC organization and vDNA trafficking during mutant MuPyV infections or inhibition of DNA synthesis. These results reveal a dynamic organization of VRC components that coordinates virus replication.
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Affiliation(s)
- Douglas K. Peters
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, Boulder, Colorado, United States of America
| | - Robert L. Garcea
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, Boulder, Colorado, United States of America
- BioFrontiers Institute, University of Colorado Boulder, Boulder, Colorado, United States of America
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Abstract
The HIV-1 capsid performs essential functions during early viral replication and is an interesting target for novel antivirals. Thus, understanding molecular and structural details of capsid function will be important for elucidating early HIV-1 (and retroviral in general) replication in relevant target cells and may also aid antiviral development. Here, we show that HIV-1 capsids stay largely intact during transport to the nucleus of infected T cells but appear to uncoat upon entry into the nucleoplasm. These results support the hypothesis that capsids protect the HIV-1 genome from cytoplasmic defense mechanisms and target the genome toward the nucleus. A protective role of the capsid could be a paradigm that also applies to other viruses. Our findings raise the question of how reverse transcription of the HIV-1 genome is accomplished in the context of the capsid structure and whether the process is completed before the capsid is uncoated at the nuclear pore. HIV-1 infects host cells by fusion at the plasma membrane, leading to cytoplasmic entry of the viral capsid encasing the genome and replication machinery. The capsid eventually needs to disassemble, but time and location of uncoating are not fully characterized and may vary depending on the host cell. To study the fate of the capsid by fluorescence and superresolution (STED) microscopy, we established an experimental system that allows discrimination of subviral structures in the cytosol from intact virions at the plasma membrane or in endosomes without genetic modification of the virus. Quantitative microscopy of infected SupT1-R5 cells revealed that the CA signal on cytosolic HIV-1 complexes corresponded to ∼50% of that found in virions at the cell surface, in agreement with dissociation of nonassembled CA molecules from entering capsids after membrane fusion. The relative amount of CA in postfusion complexes remained stable until they reached the nuclear pore complex, while subviral structures in the nucleus of infected cells lacked detectable CA. An HIV-1 variant defective in binding of the host protein cleavage and polyadenylation specificity factor 6 (CPSF6) exhibited accumulation of CA-positive subviral complexes close to the nuclear envelope without loss of infectivity; STED microscopy revealed direct association of these complexes with nuclear pores. These results support previous observations indicating capsid uncoating at the nuclear pore in infected T-cell lines. They suggest that largely intact HIV-1 capsids dock at the nuclear pore in infected SupT1-R5 cells, with CPSF6 being a facilitator of nucleoplasmic entry in this cell type, as has been observed for infected macrophages.
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Saribas AS, Coric P, Bouaziz S, Safak M. Expression of novel proteins by polyomaviruses and recent advances in the structural and functional features of agnoprotein of JC virus, BK virus, and simian virus 40. J Cell Physiol 2018; 234:8295-8315. [PMID: 30390301 DOI: 10.1002/jcp.27715] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2018] [Accepted: 10/18/2018] [Indexed: 12/30/2022]
Abstract
Polyomavirus family consists of a highly diverse group of small DNA viruses. The founding family member (MPyV) was first discovered in the newborn mouse in the late 1950s, which induces solid tumors in a wide variety of tissue types that are the epithelial and mesenchymal origin. Later, other family members were also isolated from a number of mammalian, avian and fish species. Some of these viruses significantly contributed to our current understanding of the fundamentals of modern biology such as transcription, replication, splicing, RNA editing, and cell transformation. After the discovery of first two human polyomaviruses (JC virus [JCV] and BK virus [BKV]) in the early 1970s, there has been a rapid expansion in the number of human polyomaviruses in recent years due to the availability of the new technologies and brought the present number to 14. Some of the human polyomaviruses cause considerably serious human diseases, including progressive multifocal leukoencephalopathy, polyomavirus-associated nephropathy, Merkel cell carcinoma, and trichodysplasia spinulosa. Emerging evidence suggests that the expression of the polyomavirus genome is more complex than previously thought. In addition to encoding universally expressed regulatory and structural proteins (LT-Ag, Sm t-Ag, VP1, VP2, and VP3), some polyomaviruses express additional virus-specific regulatory proteins and microRNAs. This review summarizes the recent advances in polyomavirus genome expression with respect to the new viral proteins and microRNAs other than the universally expressed ones. In addition, a special emphasis is devoted to the recent structural and functional discoveries in the field of polyomavirus agnoprotein which is expressed only by JCV, BKV, and simian virus 40 genomes.
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Affiliation(s)
- A Sami Saribas
- Laboratory of Molecular Neurovirology, Department of Neuroscience, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
| | - Pascale Coric
- Laboratoire de Cristallographie et RMN Biologiques, Université Paris Descartes, Sorbonne Paris Cité, UMR 8015 CNRS, Paris, France
| | - Serge Bouaziz
- Laboratoire de Cristallographie et RMN Biologiques, Université Paris Descartes, Sorbonne Paris Cité, UMR 8015 CNRS, Paris, France
| | - Mahmut Safak
- Laboratory of Molecular Neurovirology, Department of Neuroscience, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
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van der Meijden E, Feltkamp M. The Human Polyomavirus Middle and Alternative T-Antigens; Thoughts on Roles and Relevance to Cancer. Front Microbiol 2018; 9:398. [PMID: 29568287 PMCID: PMC5852106 DOI: 10.3389/fmicb.2018.00398] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2018] [Accepted: 02/21/2018] [Indexed: 01/08/2023] Open
Abstract
Approximately 15–20% of human cancer is related to infection, which renders them potentially preventable by antimicrobial or antiviral therapy. Human polyomaviruses (PyVs) are relevant in this regard, as illustrated by the involvement of Merkel cell polyomavirus (MCPyV) in the development of Merkel cell carcinoma. The polyomavirus Small and Large tumor antigen (ST and LT) have been extensively studied with respect to their role in oncogenesis. Recently it was shown that a number of human PyVs, including MCPyV and the trichodysplasia spinulosa polyomavirus (TSPyV), express additional T-antigens called Middle T (MT) and alternative T (ALT). ALT is encoded by ORF5, also known as the alternative T open reading frame (ALTO), which also encodes the second exon of MT, and overlaps out-of-frame with the second exon of LT. Previously, MT was considered unique for oncogenic rodent polyomaviruses, and ALT was still unknown. In this mini-review, we want to point out there are important reasons to explore the involvement of MT and ALT in human cellular transformation. First, just like their rodent equivalents, MT and ALT probably disrupt cellular pathways that control signaling and proliferation. Second, expression of the MT and ALT-encoding ORF5/ALTO characterizes a monophyletic polyomavirus clade that includes human and animal PyVs with known oncogenic potential. And third, ORF5/ALTO is subject to strong positive selection aimed specifically at a short linear motif within MT and ALT that overlaps completely with the RB-binding motif in LT. The latter suggests tight interplay between these T-antigens with possible consequences for cell transformation.
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Affiliation(s)
- Els van der Meijden
- Department of Medical Microbiology, Leiden University Medical Center, Leiden, Netherlands
| | - Mariet Feltkamp
- Department of Medical Microbiology, Leiden University Medical Center, Leiden, Netherlands
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Wu JH, Nguyen HP, Rady PL, Tyring SK. Molecular insight into the viral biology and clinical features of trichodysplasia spinulosa. Br J Dermatol 2015; 174:490-8. [PMID: 26479880 DOI: 10.1111/bjd.14239] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/14/2015] [Indexed: 02/03/2023]
Abstract
Trichodysplasia spinulosa (TS) is a disfiguring skin disease that occurs most frequently in patients receiving immunosuppressive therapies, and is thus frequently associated with organ transplantation. TS is characterized clinically by folliculocentric papular eruption, keratin spine formation and development of leonine face; and histologically by expansion of the inner root sheath epithelium and high expression of the proliferative marker Ki-67. Recent discovery of the TS-associated polyomavirus (TSPyV) and emerging studies demonstrating the role of TSPyV tumour antigens in cell proliferation pathways have opened a new corridor for research on TS. In this brief review, we summarize the clinical and histological features of TS and evaluate the current options for therapy. Furthermore, we address the viral aetiology of the disease and explore the mechanisms by which TSPyV may influence TS development and progression. As reports of TS continue to rise, clinician recognition of TS, as well as accompanying research on its underlying pathogenesis and therapeutic options, is becoming increasingly important. It is our hope that heightened clinical suspicion for TS will increase rates of diagnosis and will galvanize both molecular and clinical interest in this disease.
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Affiliation(s)
- J H Wu
- Department of Dermatology, University of Texas Health Science Center at Houston, Houston, TX, U.S.A.,Baylor College of Medicine, Houston, TX, U.S.A
| | - H P Nguyen
- Department of Dermatology, University of Texas Health Science Center at Houston, Houston, TX, U.S.A.,Baylor College of Medicine, Houston, TX, U.S.A
| | - P L Rady
- Department of Dermatology, University of Texas Health Science Center at Houston, Houston, TX, U.S.A
| | - S K Tyring
- Department of Dermatology, University of Texas Health Science Center at Houston, Houston, TX, U.S.A
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Gluschnaider U, Hertz R, Ohayon S, Smeir E, Smets M, Pikarsky E, Bar-Tana J. Long-Chain Fatty Acid Analogues Suppress Breast Tumorigenesis and Progression. Cancer Res 2014; 74:6991-7002. [DOI: 10.1158/0008-5472.can-14-0385] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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Involvement of microtubular network and its motors in productive endocytic trafficking of mouse polyomavirus. PLoS One 2014; 9:e96922. [PMID: 24810588 PMCID: PMC4014599 DOI: 10.1371/journal.pone.0096922] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2013] [Accepted: 04/14/2014] [Indexed: 12/18/2022] Open
Abstract
Infection of non-enveloped polyomaviruses depends on an intact microtubular network. Here we focus on mouse polyomavirus (MPyV). We show that the dynamics of MPyV cytoplasmic transport reflects the characteristics of microtubular motor-driven transport with bi-directional saltatory movements. In cells treated with microtubule-disrupting agents, localization of MPyV was significantly perturbed, the virus was retained at the cell periphery, mostly within membrane structures resembling multicaveolar complexes, and at later times post-infection, only a fraction of the virus was found in Rab7-positive endosomes and multivesicular bodies. Inhibition of cytoplasmic dynein-based motility by overexpression of dynamitin affected perinuclear translocation of the virus, delivery of virions to the ER and substantially reduced the numbers of infected cells, while overexpression of dominant-negative form of kinesin-1 or kinesin-2 had no significant impact on virus localization and infectivity. We also found that transport along microtubules was important for MPyV-containing endosome sequential acquisition of Rab5, Rab7 and Rab11 GTPases. However, in contrast to dominant-negative mutant of Rab7 (T22N), overexpression of dominant-negative mutant Rab11 (S25N) did not affect the virus infectivity. Altogether, our study revealed that MPyV cytoplasmic trafficking leading to productive infection bypasses recycling endosomes, does not require the function of kinesin-1 and kinesin-2, but depends on functional dynein-mediated transport along microtubules for translocation of the virions from peripheral, often caveolin-positive compartments to late endosomes and ER – a prerequisite for efficient delivery of the viral genome to the nucleus.
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Identification of an overprinting gene in Merkel cell polyomavirus provides evolutionary insight into the birth of viral genes. Proc Natl Acad Sci U S A 2013; 110:12744-9. [PMID: 23847207 DOI: 10.1073/pnas.1303526110] [Citation(s) in RCA: 129] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Many viruses use overprinting (alternate reading frame utilization) as a means to increase protein diversity in genomes severely constrained by size. However, the evolutionary steps that facilitate the de novo generation of a novel protein within an ancestral ORF have remained poorly characterized. Here, we describe the identification of an overprinting gene, expressed from an Alternate frame of the Large T Open reading frame (ALTO) in the early region of Merkel cell polyomavirus (MCPyV), the causative agent of most Merkel cell carcinomas. ALTO is expressed during, but not required for, replication of the MCPyV genome. Phylogenetic analysis reveals that ALTO is evolutionarily related to the middle T antigen of murine polyomavirus despite almost no sequence similarity. ALTO/MT arose de novo by overprinting of the second exon of T antigen in the common ancestor of a large clade of mammalian polyomaviruses. Taking advantage of the low evolutionary divergence and diverse sampling of polyomaviruses, we propose evolutionary transitions that likely gave birth to this protein. We suggest that two highly constrained regions of the large T antigen ORF provided a start codon and C-terminal hydrophobic motif necessary for cellular localization of ALTO. These two key features, together with stochastic erasure of intervening stop codons, resulted in a unique protein-coding capacity that has been preserved ever since its birth. Our study not only reveals a previously undefined protein encoded by several polyomaviruses including MCPyV, but also provides insight into de novo protein evolution.
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Pagano MA, Tibaldi E, Palù G, Brunati AM. Viral proteins and Src family kinases: Mechanisms of pathogenicity from a “liaison dangereuse”. World J Virol 2013; 2:71-78. [PMID: 24175231 PMCID: PMC3785045 DOI: 10.5501/wjv.v2.i2.71] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2012] [Revised: 01/07/2013] [Accepted: 01/24/2013] [Indexed: 02/05/2023] Open
Abstract
To complete their life cycle and spread, viruses interfere with and gain control of diverse cellular processes, this most often occurring through interaction between viral proteins (VPs) and resident protein partners. Among the latter, Src family kinases (SFKs), a class of non-receptor tyrosine kinases that contributes to the conversion of extracellular signals into intracellular signaling cascades and is involved in virtually all cellular processes, have recently emerged as critical mediators between the cell’s infrastructure and the viral demands. In this scenario, structural or ex novo synthesized VPs are able to bind to the different domains of these enzymes through specific short linear motifs present along their sequences. Proline-rich motifs displaying the conserved minimal consensus PxxP and recognizing the SFK Src homology (SH)3 domain constitute a cardinal signature for the formation of multiprotein complexes and this interaction may promote phosphorylation of VPs by SFKs, thus creating phosphotyrosine motifs that become a docking site for the SH2 domains of SFKs or other SH2 domain-bearing signaling molecules. Importantly, the formation of these assemblies also results in a change in the activity and/or location of SFKs, and these events are critical in perturbing key signaling pathways so that viruses can utilize the cell’s machinery to their own benefit. In the light of these observations, although VPs as such, especially those with enzyme activity, are still regarded as valuable targets for therapeutic strategies, multiprotein complexes composed of viral and host cell proteins are increasingly becoming objects of investigation with a view to deeply characterize the structural aspects that favor their formation and to develop new compounds able to contrast viral diseases in an alternative manner.
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Mandavia C, Sowers JR. Phosphoprotein Phosphatase PP2A Regulation of Insulin Receptor Substrate 1 and Insulin Metabolic Signaling. Cardiorenal Med 2012; 2:308-313. [PMID: 23381670 DOI: 10.1159/000343889] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/18/2012] [Accepted: 09/24/2012] [Indexed: 12/19/2022] Open
Abstract
Insulin (INS) metabolic signaling is important for normal cardiovascular and renal function as well as for exerting the classic actions of INS, such as glucose uptake in skeletal muscle tissue. There is emerging evidence that tyrosine phosphatases as well as protein kinases have important modulating roles in INS metabolic signaling in both cardiovascular and classically INS- sensitive tissues. For example, increases in phosphatase activity may partially explain how angiotensin II and aldosterone attenuate activation of the INS receptor substrate protein 1 (IRS-1)-phosphatidylinositol 3 kinase-protein kinase B pathway, thereby promoting INS resistance. On the other hand, phosphatase activation may also exert beneficial and cardiovascular protective effects in conditions such as overnutrition by blocking serine phosphorylation of IRS-1, thereby improving downstream INS metabolic signaling. Both the beneficial and the detrimental effects exerted by the activation of phosphatases will be covered in this report.
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Affiliation(s)
- Chirag Mandavia
- Department of Internal Medicine, University of Missouri-Columbia School of Medicine, Columbia, Mo., USA
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